The present invention relates to methods for controlling glycosylation of proteins and compositions and methods relating thereto. The invention further relates to methods of decreasing glycosylation and/or the glycan site occupancy of glycoproteins thereby affecting their biological characteristics and/or functions. The invention also relates to compositions produced using the methods of the invention to control glycosylation and uses thereof.
Proteins and polypeptides have become increasingly important therapeutic agents. In most cases, these proteins and polypeptides are produced in cell culture, from cells that have been engineered and/or selected to produce unusually high levels of the particular protein or polypeptide of interest. Control and optimization of cell culture conditions is critically important for successful commercial production of proteins and polypeptides.
Many proteins and polypeptides produced in cell culture are glycoproteins that contain covalently linked carbohydrate structures including oligosaccharide chains. These oligosaccharide chains are linked to the protein in the endoplasmic reticulum and the Golgi apparatus via either N-linkages or O-linkages. The oligosaccharide chains may comprise a significant portion of the mass of the glycoprotein. The oligosaccharide chains are thought to play key roles in the function of the glycoprotein including facilitating correct folding of the glycoprotein, mediating protein-protein interactions, conferring stability, conferring advantageous pharmacodynamic and/or pharmacokinetic properties, inhibiting proteolytic digestion, targeting the glycoprotein to the proper secretory pathway and targeting the glycoprotein to a particular organ or organs.
Generally, N-linked oligosaccharide chains are added to the nascent, translocating protein in the lumen of the endoplasmic reticulum (see Alberts et al., 1994, In: Molecular Biology of the Cell, incorporated herein by reference). The oligosaccharide is added to the amino group on the side chain of an asparagine residue contained within the target consensus sequence of Asn-X-Ser/Thr, where X may be any amino acid except proline. The initial oligosaccharide chain is usually trimmed by specific glycosidase enzymes in the endoplasmic reticulum, resulting in a short, branched core oligosaccharide composed of two N-acetylglucosamine and three mannose residues.
After initial processing in the endoplasmic reticulum, the glycoprotein is shuttled via small vesicles to the Golgi apparatus, where the oligosaccharide chain undergoes further processing before being secreted to the cell surface. The trimmed N-linked oligosaccharide chain may be modified by the addition of several mannose residues, resulting in a high-mannose oligosaccharide. Alternatively, one or more monosaccharides units of N-acetylglucosamine may be added to the core mannose subunits to form complex oligosaccharides. Galactose may be added to the N-acetylglucosamine subunits, and sialic acid subunits may be added to the galactose subunits, resulting in chains that terminate with either a sialic acid, a galactose or an N-acetylglucosamine residue. Additionally, a fucose residue may be added to an N-acetylglucosamine residue of the core oligosaccharide. Each of these additions is catalyzed by specific glycosyl transferases.
In addition to being modified by the N-linked glycosylation pathway, glycoproteins may also be modified by the addition of O-linked oligosaccharide chains to specific serine or threonine residues as they are processed in the Golgi apparatus. The residues of an O-linked oligosaccharide are added one at a time and the addition of each residue is catalyzed by a specific enzyme. In contrast to N-linked glycosylation, the consensus amino acid sequence for O-linked glycosylation is less well defined.
Glycosylation of proteins, especially immunoglobulins, has been shown to have significant effects on their biological functions. For instance, for immunoglobulins, glycosylation has been demonstrated to affect effector functions, structural stability, and rate of secretion from antibody-producing cells (Leatherbarrow et al., 1985, Mol. Immunol. 22:407). The carbohydrate groups responsible for these properties are generally attached to the constant (C) regions of the antibodies. For example, glycosylation of IgG at asparagine 297 in the CH2 domain is required for full capacity of IgG to activate the classical pathway of complement-dependent cytolysis (Tao and Morrison, 1989, J. Immunol. 143:2595). Glycosylation of IgM at asparagine 402 in the CH3 domain is necessary for proper assembly and cytolytic activity of the antibody (Muraoka and Shulman, 1989, J. Immunol. 142:695). Removal of glycosylation sites as positions 162 and 419 in the CH1 and CH3 domains of an IgA antibody led to intracellular degradation and at least 90% inhibition of secretion (Taylor and Wall, 1988, Mol. Cell. Biol. 8:4197).
Glycosylation of immunoglobulins in the variable (V) region, comprising the antigen binding site, has also been observed. Sox and Hood (1970, Proc. Natl. Acad. Sci. USA 66:975), reported that about 20% of human antibodies are glycosylated in the V region. Glycosylation of the V domain is believed to arise from fortuitous occurrences of the N-linked glycosylation signal Asn-Xaa-Ser/Thr in the V region sequence and has not been recognized in the art as playing an important role in immunoglobulin function.
More recently, it has been demonstrated that glycosylation at CDR2 of the heavy chain, in the antigen binding site, of a murine antibody specific for alpha-(1-6)dextran increases its affinity for dextran (Wallick et al., 1988, J. Exp. Med. 168:1099; and Wright et al., 1991, EMBO J. 10:2717). It has also been demonstrated that mutation to remove a glycosylation site within the antigen binding site of an anti-CD33 antibody, more specifically, removal of an N-linked glycosylation site within a framework region of the V domain of the antibody M195, enhanced antibody binding with the antigen (U.S. Pat. No. 6,350,861, to Co et al.). However, the N-linked glycosylation site present in the Fc portion of the antibody was not removed and was, preferably, glycosylated to maintain the effector function of the molecule.
Additionally, glycolysis-inhibiting substances have been added to cell culture medium to reduce accumulation of the metabolic waste product lactate thereby increasing cell viability and protein production of antibodies. See, e.g., International Patent Application No. PCT/US2007/083473 now published as WO 2008/055260 on May 8, 2008 (addition of glycolysis inhibiting compound to cell culture reduced lactate concentration and increased production of antibody specific for growth and differentiation factor 8 (GDF-8)). While glycolysis-inhibition may also have affected the level of protein glycosylation, the effect of the inhibitor on protein glycosylation was not assessed or even discussed. Moreover, the anti-GDF-8 antibodies produced in cell culture did not comprise any potential glycosylation site in the antigen binding site (see WO 2008/055260 at page 49, paragraph 170, citing Veldman et al., U.S. Patent Publication No. 2004/0142382, describing the anti-GDF-8 antibodies Myo22, Myo28 and Myo29). Thus, even assuming the glycosylation of the anti-GDF-8 antibody at the heavy chain constant domain was affected under the culture conditions disclosed, no effect of altered glycosylation of the antigen binding site could have been assessed since it appears that the antibodies produced lacked a potential glycosylation site in the antigen binding region (e.g., the V domain).
A major problem with protein therapeutics has been reduced or low affinity for the ligand or antigen. Loss of or decreased affinity is highly undesirable, and requires that more of the therapeutic protein will have to be injected into the patient, at higher cost and greater risk of adverse effects. Even more critically, protein with reduced affinity may have decreased biological functions, such as complement lysis, antibody-dependent cellular cytotoxicity, and virus neutralization. Further, the protein may have decreased antagonist function by way of decreased competition between the antigen or binding partner and the therapeutic protein compared with the endogenous ligand or binding partner the interaction of which is sought to be inhibited. For example, the loss of affinity in the partially humanized antibody HUVHCAMP may have caused it to lose all ability to mediate complement lysis (see, Riechmann et al., 1988, Nature, 332, 323-327, at Table 1).
Further, given the unpredictability of protein conformation, mutation of even a single amino acid, especially at the antigen binding site or ligand binding site of the protein can have drastic effects on the biological activity of a potential therapeutic protein.
Thus, there exists a need in the art for therapeutic proteins that have an altered affinity for a ligand or, in the case of an antibody, an antigen, particularly an increased affinity and/or increased specificity for an antigen or ligand, and, desirably, potentially lower immunogenicity and improved effector function conferred by naturally-occurring constant region glycosylation. Alternatively, there exists a need for a therapeutic protein, especially an antibody or an Fc fusion protein comprising an immunoglobulin constant region, or a portion thereof, having improved binding affinity and/or specificity for an antigen or a ligand, but which has decreased or no effector function mediated by glycosylation of the glycosylation site present in the antibody heavy chain constant region. There is a further need to inhibit or remove glycosylation of a therapeutic protein antigen or ligand binding site without the need to introduce any mutation into the amino acid sequence of the antigen or ligand binding site. Thus, there exists a need in the art for methods to increase the efficacy and reduce the required doses of immunoglobulins and other proteins of therapeutic importance, and for therapeutic proteins produced by such methods, and the present invention meets these needs without requiring the introduction of any mutation into the antigen binding site of an antibody or the ligand binding site of a therapeutic protein.
Despite the importance of therapeutic glycoproteins and the advances in cell culture processes, there is an unmet need for novel processes for producing such proteins under conditions where glycosylation of the ligand binding site may be controlled without requiring the introduction of an amino acid mutation into such a critical portion of the protein. The present invention meets this need.